The effect on clinical results of adding recombinant LH in late phase of ovarian stimulation of patients with repeated implantation failure: a pilot study.

OBJECTIVE: To evaluate the efficacy of recombinant LH (r-LH) addition in the late phase of ovarian stimulation in patients with repeated implantation failure (RIF). PATIENTS AND METHODS: 66 infertile couples undergoing ICSI treatment due to male factor were allocated to group A (33) and to group B (33). Group A (29 subjects) received recombinant FSH (r-FSH) supplemented by r-LH in the late follicular phase starting the same day of GnRH-antagonist (GnRH-ant) administration, and group B (32 subjects) received r-FSH alone. All patients were stimulated with a GnRH-ant flexible protocol starting r-FSH on day 2 of a spontaneous or induced cycle. hCG (10000 IU) was administered by intramuscular route when at least 2 follicles reached 18 mm in diameter. RESULTS: Metaphase II (MII) oocytes with cytoplasmic maturation showed a significant difference in the r-LH group (89.02%) compared to the one with FSH alone (81.15%) (p < 0.01). The number of positive pregnancy test, 14 (48.3%) and 8 (25%), was significantly greater in the r-LH group compared to the group treated with r-FSH alone (p < 0.03). The number of gestational sacs was 20 in the r-LH group vs. 9 in the r-FSH group (p < 0.001). The implantation rate was significantly higher in the r-LH group compared to the r-FSH only group (19% vs. 7% respectively; p < 0.01). Also, a lower abortion rate was found in the r-LH group (21% vs. 37.5% respectively - p < 0.01). CONCLUSIONS: Ovarian stimulation should be personalized because it seems that some subgroups of patients, like those with RIF, reach a better clinical outcome with the addition of r-LH in the advanced follicular phase stimulation.

The use of r-hFSH in treatment of idiopathic male factor infertility before ICSI.

OBJECTIVE: To evaluate the efficacy of pre-treatment of idiopathic oligozoospermic patients with r-hFSH to improve the clinical results of ICSI. PATIENTS AND METHODS: 82 infertile couples due to male factor who attended our center were included in the study. Thirty-six were randomized to the treatment group (group A) and forty-six to the control group (group B). The male partners in group A were treated with recombinant human FSH (r-hFSH; Gonal F®) 150 IU subcutaneously three times a week for a 3-months period. The control group (group B) did not receive any treatment. After the treatment couples of both groups underwent a cycle of ICSI. RESULTS: The fertilization rate was comparable in both groups. However, in the treatment group (group A), the clinical pregnancy rate was significantly higher (42%) compared to the control group (group B) (20%) (p < 0.02). Also, the implantation rate was significantly higher in treatment group (26%) compared to the control (15%) (p < 0.04). Miscarriage rate was lower (15.7%) in the treatment group than in the control (43.7%), and this difference was statistically significant (p < 0.05). CONCLUSIONS: Treatment of idiopathic male factor infertility with r-hFSH before ICSI improves clinical pregnancy rate, increases implantation rate and decreases the early pregnancy loss.

Decentralized diagnostics based on a distributed micro-genetic algorithm for transducer networks monitoring large experimental systems.

Evolutionary approach to centralized multiple-faults diagnostics is extended to distributed transducer networks monitoring large experimental systems. Given a set of anomalies detected by the transducers, each instance of the multiple-fault problem is formulated as several parallel communicating sub-tasks running on different transducers, and thus solved one-by-one on spatially separated parallel processes. A micro-genetic algorithm merges evaluation time efficiency, arising from a small-size population distributed on parallel-synchronized processors, with the effectiveness of centralized evolutionary techniques due to optimal mix of exploitation and exploration. In this way, holistic view and effectiveness advantages of evolutionary global diagnostics are combined with reliability and efficiency benefits of distributed parallel architectures. The proposed approach was validated both (i) by simulation at CERN, on a case study of a cold box for enhancing the cryogeny diagnostics of the Large Hadron Collider, and (ii) by experiments, under the framework of the industrial research project MONDIEVOB (Building Remote Monitoring and Evolutionary Diagnostics), co-funded by EU and the company Del Bo srl, Napoli, Italy.

Pineal-adrenal interactions in domestic male pigeon exposed to variable circadian light regimes and exogenous melatonin.

OBJECTIVE: This study was aimed to see the histophysiological changes of the pineal and adrenal glands under altered photoperiodic conditions due to the administration of melatonin in the male domestic pigeon, Columba livia Gmelin. METHODS: Young adult male domestic pigeons were exposed to long photoperiod (LP; 20 hr light: 4 hr dark), short photoperiod (SP; 4 hr light: 20 hr dark) exogenous melatonin (MEL; 20 microg/100 g body weight/day) was administered for 60 days in the primary breeding and regressive phase. At the end of experiment, adrenal and pineal glands were quickly dissected and processed for histology, ultrastructure study, biochemical, histochemical and immunohistochemical analysis. RESULTS: In the primary breeding phase, the number of mitochondria and rough endoplasmic reticulum were increased in the adrenal gland in MEL-LP group, while lipid granules were also increased in the subcapsular zone. In MEL-SP treated group, however, the number of mitochondria decreased. Pinealocytes were increased in size and well-developed Golgi complexes were present near the cell nucleus after induction with MEL-LP treatment during the primary breeding phase. No remarkable changes were noticed in the number of mitochondria. In the regressive phase-I, the adrenocortical cytoplasm showed similar morphological features both in MEL-LP treatment and control groups. In pinealocytes, few rough endoplasmic reticulum and lipid droplets and moderate number of mitochondria were present. In MEL-SP treatment, increased number of mitochondria in the adrenocortical cells and decreased nuclear diameter of the pinealocytes were noticed. Few mitochondria were observed within the pinealocyte cytoplasm. Side chain cleavage enzyme (immunocytochemical) activity was increased in the subcapsular zone in MEL-LP treated group. CONCLUSIONS: The present data indicates that the changes in pineal and adrenocortical tissue histophysiology might be due to melatonin rhythm and light/dark regime which act as a modulator in the male domestic pigeon.

Serogroup distribution and virulence characteristics of sorbitol-negative Escherichia coli from food and cattle stool.

AIMS: To (i) study the serogroup distribution and virulence characteristics of non-sorbitol-fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re-examine the true sorbitol and beta-D-glucuronidase (GUD) reactions of sorbitol-negative (Sor(-)) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. METHODS AND RESULTS: One hundred and thirty Sor(-)E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite-supplemented SMAC (CT-SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O-rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O-rough and OUT strains were enterohaemolytic. Further, 39.2% and 63.1% of Sor(-) isolates from CT-SMAC fermented sorbitol in phenol red broth and hydrolysed 4-methylumbelliferyl-beta-D-glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O-rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. CONCLUSIONS: Sor(-)E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14.6% of the isolates. Many of these nonclinical Sor(-) strains were potentially pathogenic. Nearly 39% of these Sor(-)E. coli from CT-SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. SIGNIFICANCE AND IMPACTS OF THE STUDY: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.

Recombinant HCG for triggering ovulation increases the rate of mature oocytes in women treated for ICSI.

PURPOSE: To conduct a prospective randomized study in order to investigate the effect of recombinant HCG (rHCG) on oocyte nuclear and cytoplasm maturity compared to urinary HCG (uHCG), for inducing ovulation in women treated with ICSI for male factor infertility. MATERIALS AND METHODS: We compared 89 patients randomly assigned to one of the two study groups. Group A consisted of 42 women who received a subcutaneous (s.c.) injection of 250 microg rHCG and group B consisted of 47 patients receiving an intramuscular (i.m.) injection of 10,000 IU uHCG. RESULTS: Patients treated with rHCG showed a rate of metaphase II oocytes, a number of metaphase II oocytes with mature cytoplasm and a rate of metaphase II oocytes with mature cytoplasm calculated from total MII oocytes statistically higher than in patients treated with uHCG. However this differences were not associated with a significantly better clinical outcome. CONCLUSION: Our data show that in women treated with ICSI for male factor infertility, rHCG increases the rate of metaphase II oocytes, the number and the rate of MII oocytes with mature cytoplasm compared to uHCG. A larger study comparing transfer cycles of embryos all derived from oocytes with mature cytoplasm and transfer cycles of embryos all derived from oocytes with immature cytoplasm may be needed to clarify clinical correlations.

Microbiological quality of finfish and shellfish with special reference to shiga toxin-producing Escherichia coli O157.

The objective of this study was to determine the microbiological quality of fish and shellfish from Kolkata, India, with special emphasis on E. coli O157. Fresh and ice-preserved Labeo rohita, Catla catla, Cirrhinus mrigala, Oreochromis mossambica, Heteropneustesfossilis, Clarias batrachus, and Penaeus monodon were examined for total heterotrophic bacteria and coliform loads and presence of E. coli and E. coli serotype O157 by culture method. While the total plate count of bacteria was within acceptable or marginally acceptable limits for most samples, fishes were contaminated with coliforms, including E. coli, indicating poor hygiene and sanitary conditions. Although E. coli O157 could not be detected, a few samples were contaminated with non-O157 serotypes of enterohaemolysin- and Shiga toxin-producing E. coli, raising public health concern.

Occurrence, virulence characteristics and antimicrobial resistance of Escherichia coli O157 in slaughtered cattle and diarrhoeic calves in West Bengal, India.

AIMS: (i) To study the occurrence of Escherichia coli serotype O157 in cattle stool in West Bengal, India, and (ii) the virulence properties and antimicrobial resistance of the E. coli isolates. METHODS AND RESULTS: Following enrichment in modified EC broth and plating onto HiCrome MS.O157 agar, a total of 14 strains of E. coli serotype O157 was isolated from faecal samples from two (2.04%) slaughtered cattle and six (7.59%) diarrhoeic calves. By multiplex PCR, Shiga toxin genes were detected in all the isolates. The enterohaemolysin phenotype was found in all, but one strain. Among 14 strains, ten were resistant to at least one of the antimicrobial agents tested. Multiple antibiotic resistance was frequent. CONCLUSIONS: The study showed that occurrence of Shiga toxin-producing and multiple antibiotic-resistant E. coli O157 among cattle population in this region of India is significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering routine human contacts with cattle, a large human population in this region may be at risk for exposure to Shiga toxin-producing E. coli O157.

Experimental results on the recognition of embryos in human assisted reproduction.

The recognition of embryos suitable for transfer in human assisted reproduction is important, and there is evidence that the morphology of the cells may influence the results achievable. A procedure for this recognition problem has been formulated based on morphological attributes of the images of the embryos, and it is therefore useful to compare the recognition of experts with that of a machine programme. The aim of this paper is to compare the precision in the recognition of viable embryos by a group of experts to that of a machine recognition procedure, both for a basic set of embryos and a blind set. Experts were asked to classify the images of 249 embryos transferred to 73 patients, indicated as the training set and another set of 103 embryos transferred to 35 patients called the blind set. A machine programme was used for the same classification. For all the experts the results were statistically not significantly different from independence, which means that viable embryos are not recognized as such for both data sets. Instead, the machine algorithm recognizes in a statistically significant way, the membership class for the embryos submitted. Cell morphology is important for IVF, but differences do not appear to be discernable by the senses, clinical insight, experience and/or training, while classification by machine methods provides more accurate results, which could be improved by enlarging the training set.

Artificial kidney: status of the art and new perspectives.

Extracorporeal dialysis was first performed in 1943 and has become a routine for End Stage Renal Patients from the early sixties. In the last 30 years researchers have focused on biocompatibility of artificial materials and optimisation of removal of uremic toxins by the membrane as in the long term treatment many complications like amylodosis heart and bone lesions, accelerated amyloidosis and immune system failure can occur. From this point of view high flux dialytic membranes are currently considered more biocompatible therefore being able to prevent such diseases.

Antifertility effects of methylmethane sulphonate on male reproductive organs of wild Indian house rat: histological and histochemical characteristics.

OBJECTIVE: To study the effects of methylmethane sulphonate (CH3OSO2CH3), on the testicular tissue of the adult wild Indian house rat (Rattus rattus). METHODS: A single intraperitoneal dose of methylmethane sulphonate (20 mg/kg) was administered and the effects were observed 2, 7, 15, 30 and 45 days later. RESULTS: Significant changes of the body, testes and accessory reproductive organs weight and a major depletion of the relative percentages of the spermatid and spermatozoa were noticed at 2, 7, 15 and 30 days after treatment. Gradual decrease in the seminiferous tubular area and Sertoli cell nuclear diameter was observed at 7, 15 and 30 days of treatment groups. The sperm population and sperm morphological abnormalities were also noticed in these three groups. Histochemical studies clearly revealed that the intensity of staining of the acid and alkaline phosphatase within 7 and 15 days after treatment was decreased, while the quantity of lipid materials was increased especially on the 2nd and 7th day after treatment. However, no significant changes were noticed in the delta5-3beta-HSDH and 17beta-HSDH enzymatic activity in the treated animals. CONCLUSION: These observations showed the antispermatogenic activity of methylmethane sulphonate on the testicular tissues and various accessory reproductive organs in the wild Indian house rat (Rattus rattus).

Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil.

3,4-Dihydroxyphenylethanol (DOPET) is the major o-diphenol detectable in extra virgin olive oil, either in free or esterified form. Despite its relevant biological effects, mainly related to its antioxidant properties, little data have been reported so far on its toxicity and metabolism. The aim of the present work is to evaluate DOPET toxicity and to investigate its molecular pharmacokinetics by using the (14)C-labeled diphenol. When orally administered to rats, the molecule does not show appreciable toxicity up to 2 g/kg b.wt. To identify and quantify its metabolites, [(14)C]DOPET has been synthesized and intravenously injected in rats. The pharmacokinetic analysis indicates a fast and extensive uptake of the molecule by the organs and tissues investigated, with a preferential renal uptake. Moreover, 90% of the administered radioactivity is excreted in urine collected up to 5 h after injection, and about 5% is detectable in feces and gastrointestinal content. The characterization of the labeled metabolites, extracted from the organs and urine, has been performed by high-pressure liquid chromatography analysis. In all the investigated tissues, DOPET is enzymatically converted in four oxidized and/or methylated derivatives. Moreover, a significant fraction of total radioactivity is associated with the sulfo-conjugated forms, which also represent the major urinary excretion products. On the basis of the reported results, an intracellular metabolic pathway of exogenously administered DOPET, implying the involvement of catechol-O-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, and phenolsulfotransferase, has been proposed.

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques.

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

A prospective randomized study comparing intramuscular progesterone and 17alpha-hydroxyprogesterone caproate in patients undergoing in vitro fertilization-embryo transfer cycles.

OBJECTIVE: To compare the effectiveness of i.m. P and 17alpha-hydroxyprogesterone caproate (17-HPC) for luteal phase support, in patients undergoing IVF-ET cycles. DESIGN: Prospective, randomized study. SETTING: Patients undergoing IVF-ET in our Centers. PATIENT(S): The inclusion criteria were the use of GnRH down-regulation and aged <40 years. INTERVENTION(S): A total of 300 cycles were randomly treated with either 17-HPC (341 mg every 3 days) or P (50 mg daily). MAIN OUTCOME MEASURE(S): The outcomes of IVF in both study groups were evaluated for biochemical pregnancy, miscarriage, clinical pregnancy, and ongoing pregnancy. RESULT(S): No difference was found in the main outcome parameters considered. CONCLUSION(S): Although the results of the study encourage the use of 17-HPC for luteal phase support in patients undergoing IVF-ET program, more studies are necessary to support the hypothesis that it can replace i.m. P-in-oil.

Histophysiological changes of the testicular tissue due to busulphan administration in the wild Indian house rat (Rattus rattus).

The results of the present study indicate the antispermatogenic activity of Busulphan or Myleran (1,4-dimethane-sulphonoxy butane) on the testicular tissue of adult male Indian house rat, Rattus rattus. Single oral dose of Busulphan (10 mg/Kg body weight) was administered and its activity was noticed at 10, 40, 70 and 100 days of posttreated animals. Histological observation and quantitative histological study indicates no major alteration in the relative percentages of primary spermatocytes, spermatid and Sertoli cells at 10 days of posttreatment. But there was a gradual decrease in the seminiferous tubular diameter at 40 and 70 days of post treated groups. However, the Leydig and Sertoli cells morphology and number remained normal in all the treatment groups. At 40 days, the normal cellular associations in all the tubules were disrupted. The tubules constituted only spermatogonia, Sertoli cells and some zygotene spermatocytes. At 70 days, repopulation of Type A, Type B spermatogonia, resting and zygotene spermatocytes occurred at this stage. The tubules were still devoid of pachytene spermatocytes, spermatid and spermatozoa. At 100 days, active spermatogenesis was observed in majority of the tubules. The various types of germ cell population were regaining towards normalcy. Histochemical studies clearly revealed that due to busulphan administration there was no major alteration in the intensities of some key enzymes (i.e. delta5 3beta-HSDH and 17beta-HSDH) involved in the biosynthesis of steroid hormones. Only the acid phosphatase activity was slightly depressed within the 40th and 70th days of posttreatment. Sudanophilic lipid materials increased in the interstitium of all the busulphan post treated groups. The changes which were noticed due to busulphan treatment regained normalcy at 100 days of post treated animals. The mode of action of Busulphan on the testicular tissue of adult Indian house rat (Rattus rattus) has been pointed out and discussed.

Intra- and post-dialytic platelet activation and PDGF-AB release: cellulose diacetate vs polysulfone membranes.

BACKGROUND: During haemodialysis the blood-membrane contact causes a release of platelet granule content, which contains platelet-derived growth factor AB (PDGF-AB). In view of the potential role of this in altering biocompatibility during haemodialysis, we evaluated the intra- and post-dialytic changes in PDGF-AB serum levels during haemodialysis sessions performed with cellulose diacetate (CDA) and polysulfone (PS) membranes respectively. METHODS: PDGF-AB, platelet factor 4 (PF4), beta thromboglobulin (betaTG), and mean platelet volume (MPV) levels were determined in 30 patients, each of whom underwent six dialysis sessions: three with a CDA and three with a PS membrane. Blood samples were taken at times 0, 15, 30, 120, 180, and 240 min during dialysis and at 1, 4, and 20 h after the end of the session. Statistical analysis was performed using a one-way ANOVA and Student's t test. RESULTS: PDGF-AB at 15 min was increased to +41+/-9% with CDA vs +20+/-5% with PS (P<0.001) from the T0 values, and at 120 min it was +19+/-8% with CDA vs -25+/-9% with PS (P<0.001) from T0 levels. At 240 min it was +95+/-14% with CDA vs +49+/-15% with PS (P<0.001) from the T0 values, returning to basal only 20 h after the end of the session. betaTG at 15 min was +60+/-8% for CDA vs +24+/-7.5% for PS (P<0.001) from the T0 values. PF4 showed a similar trend to betaTG. MPV at 30 min from the start of dialysis was 7.4+/-0.3 fl with CDA and 8+/-0.3 fl with PS (P<0.001), and at 240 min MPV was 7.9+/-0.3 fl with CDA and 8.4+/-0.3 fl with PS (P<0.001). CONCLUSIONS: Platelet activation and platelet release reactions are lower with PS than with CDA membranes. PDGF-AB, released during and after dialysis, represents a clear biocompatibility marker. Its slow return to basal values and its action on vascular cells make it a potential risk factor for atherosclerosis in uraemic patients.

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Use of intramuscular progesterone versus intravenous albumin for the prevention of ovarian hyperstimulation syndrome.

This study was designed to compare the effectiveness of intramuscular progesterone with that of intravenous albumin in the prevention of ovarian hyperstimulation syndrome (OHSS). Ninety-six patients at high risk to develop OHSS (estradiol concentration >9, 000 pmol/l on the day of hCG administration and over 20 follicles of a diameter larger than 14 mm observed by transvaginal ultrasonography) and undergoing in vitro fertilization-embryo transfer were enrolled. They were randomly treated with intramuscular progesterone (200 mg/day) or 100 ml of 20% intravenous albumin in order to estimate the difference in the incidence of OHSS. A significant difference in the incidence of moderate OHSS and no cases of severe OHSS were observed between the groups. Our data show the effectiveness in preventing OHSS with high doses of progesterone.